Immunological and molecular diagnostic assays play a critical role both in the research and clinical fields. Often it is necessary to perform assays for a panel of multiple targets to gain meaningful or bird's-eye view results to facilitate research or clinical decision-making. This is particularly true in the era of genomics and proteomics, where an abundance of genetic markers and/or biomarkers are thought to influence or be predictive of particular disease states. In theory, assay of multiple targets can be accomplished by testing each target separately in parallel or sequentially in different reaction vessels (i.e., multiple singleplexing). However, not only are assays adopting a singleplexing strategy often cumbersome, but they also typically required large sample volumes, especially when the targets to be analyzed are large in number.
A multiplex assay simultaneously measures multiple analytes (two or more) in a single assay. Multiplex assays are commonly used in high-throughput screening settings, where many specimens can be analyzed at once. It is the ability to assay many analytes simultaneously and many specimens in parallel that is the hallmark of multiplex assays and is the reason that such assays have become a powerful tool in fields ranging from drug discovery to functional genomics to clinical diagnostics. In contrast to singleplexing, by combining all targets in the same reaction vessel, the assay is much less cumbersome and much easier to perform since only one reaction vessel is handled per sample. The required test samples can thus be dramatically reduced in volume, which is especially important when samples (e.g., tumor tissues, cerebral spinal fluid, or bone marrow) are difficult and/or invasive to retrieve in large quantities. Equally important is the fact that the reagent cost can be decreased and assay throughput increased drastically.
Many technologies for multiplex detection are available, including fluorescent-coded beads, barcoded magnetic beads, etc. Traditionally, all these suspension array beads are utilized for performing the actual bioassays. In these bead-based multiplex assay systems, there are two identification systems for every bead in the assay. One system is for the identification of the capture agent attached to the surface of the beads while the second identification system is used to indicate the presence or quantity of the analyte that binds to the particular capture agent. The Luminex technology is an example of a bead-based multiplex detection system centered on latex beads that have two different fluorophores associated with any given bead. The first fluorescent dye is injected into the beads during the latex polymerization process and is used to reveal the identity of the beads (i.e. the identification of the capture agent associated with the bead). The second fluorophore is conjugated to an analyte binder introduced to the beads when there is an analyte molecule captured by the bead-linked analyte capture agent. In other bead-based assays, the first identification system can be replaced by systems other than those which are fluorescence-based. For example, in Applied Biocode's BMB system, the first identification system is replaced by a barcode.
In spite of these advances, there remains a need for methods and systems utilizing individually identifiable beads for use in multiplex high-throughput assays that not only ensure high precision and reproducibility of experimental results, but which also are capable of performing other functions related to information and data storage that are not directly related to the assays per se.
Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.